Characterization Of Outer Membrane Proteins Research Articles

In silico designing and characterization of outer membrane protein K (OmpK) from Vibrio anguillarum and its expression in Nicotiana tabacum for the development of a plant-based vaccine against fish vibriosis

Vibriosis is caused by Vibrio anguillarum in various species of aquaculture. A novel, secure, and stable vaccine is needed to eradicate vibriosis. Here, for reverse vaccinology and plant-based expression, the outer membrane protein K (OmpK) of V. anguillarum was chosen due to its conserved nature in all Vibrio species. OmpK, an ideal vaccine candidate against vibriosis, demonstrated immunogenic, non-allergic, and non-toxic behavior by using various bioinformatics tools. Docking showed the interaction of the OmpK model with TLR-5. In comparison to costly platforms, plants can be used as alternative and economic bio-factories to produce vaccine antigens. We expressed OmpK antigen in Nicotiana tabacum using Agrobacterium-mediated transformation. The expression vector was constructed using Gateway® cloning. Transgene integration was verified by polymerase chain reaction (PCR), and the copy number via qRT-PCR, which showed two copies of transgenes. Western blotting detected monomeric form of OmpK protein. The total soluble protein (TSP) fraction of OmpK was equivalent to 0.38% as detected by ELISA. Mice and fish were immunized with plant-derived OmpK antigen, which showed a significantly high level of anti-OmpK antibodies. The present study is the first report of OmpK antigen expression in higher plants for the potential use as vaccine in aquaculture against vibriosis, which could provide protection against multiple Vibrio species due to the conserved nature OmpK antigen.

Syphilis vaccine: challenges, controversies and opportunities.

Syphilis is a sexually or vertically (mother to fetus) transmitted disease caused by the infection of Treponema pallidum subspecie pallidum (TPA). The incidence of syphilis has increased over the past years despite the fact that this bacterium is an obligate human pathogen, the infection route is well known, and the disease can be successfully treated with penicillin. As complementary measures to preventive campaigns and early treatment of infected individuals, development of a syphilis vaccine may be crucial for controlling disease spread and/or severity, particularly in countries where the effectiveness of the aforementioned measures is limited. In the last century, several vaccine prototypes have been tested in preclinical studies, mainly in rabbits. While none of them provided protection against infection, some prototypes prevented bacteria from disseminating to distal organs, attenuated lesion development, and accelerated their healing. In spite of these promising results, there is still some controversy regarding the identification of vaccine candidates and the characteristics of a syphilis-protective immune response. In this review, we describe what is known about TPA immune response, and the main mechanisms used by this pathogen to evade it. Moreover, we emphasize the importance of integrating this knowledge, in conjunction with the characterization of outer membrane proteins (OMPs), to expedite the development of a syphilis vaccine that can protect against TPA infection.

B-Cell Epitope Mapping of TprC and TprD Variants of Treponema pallidum Subspecies Informs Vaccine Development for Human Treponematoses.

Several recent studies have focused on the identification, functional analysis, and structural characterization of outer membrane proteins (OMPs) of Treponema pallidum (Tp). The Tp species encompasses the highly related pallidum, pertenue, and endemicum subspecies of this pathogen, known to be the causative agents of syphilis, yaws, and bejel, respectively. These studies highlighted the importance of identifying surface-exposed OMP regions and the identification of B-cell epitopes that could be protective and used in vaccine development efforts. We previously reported that the TprC and TprD OMPs of Tp are predicted to contain external loops scattered throughout the entire length of the proteins, several of which show a low degree of sequence variability among strains and subspecies. In this study, these models were corroborated using AlphaFold2, a state-of-the-art protein structure modeling software. Here, we identified B-cell epitopes across the full-length TprC and TprD variants using the Geysan pepscan mapping approach with antisera from rabbits infected with syphilis, yaws, and bejel strains and from animals immunized with refolded recombinant TprC proteins from three syphilis strains. Our results show that the humoral response is primarily directed to sequences predicted to be on surface-exposed loops of TprC and TprD proteins, and that the magnitude of the humoral response to individual epitopes differs among animals infected with various syphilis strains and Tp subspecies. Rather than exhibiting strain-specificity, antisera showed various degrees of cross-reactivity with variant sequences from other strains. The data support the further exploration of TprC and TprD as vaccine candidates.

Atomic-level characterization of outer membrane proteins from Yersinia pestis in near-native membrane environments

The virulence factors Adhesion invasion locus (Ail) and Plasminogen Activator (Pla) from Yersinia pestis are involved in enhanced bacterial dissemination and serum survival within humans. The localization of these virulence factors to the bacterial outer membrane, which is unique due to its high asymmetry, subjects the two proteins to functional regulation by components of the outer membrane, such as lipopolysaccharide (LPS). Prior structural and functional works have highlighted the importance of these protein-membrane interactions for both binding and proteolysis of known human serum factors essential for the high virulence of Y.

Reverse vaccinology approach for the identification and characterization of outer membrane proteins of Shigella flexneri as potential cellular- and antibody-dependent vaccine candidates.

PurposeIn the developing world, bacillary dysentery is one of the most common communicable diarrheal infections. There are approximately 169 million cases of shigellosis reported worldwide. The disease is transmitted by a group of Gram-negative intracellular enterobacteria known as Shigella flexneri, S. sonnei, S. dysenteriae, and S. boydii. Conventional treatment regimens for Shigella have been less effective due to the development of resistant strains against antibiotics. Therefore, an effective vaccine for the long term control of Shigella transmission is urgently needed.Materials and MethodsIn this study, a reverse vaccinology approach was employed to identify most conserved and immunogenic outer membrane proteins (OMPs) of S. flexneri 2a.ResultsFive OMPs including fepA, ompC, nlpD_1, tolC, and nlpD_2 were identified as potential vaccine candidates. Protein-protein interactions analysis using STRING software (https://string-db.org/) revealed that five of these OMPs may potentially interact with other intracellular proteins which are involved in beta-lactam resistance pathway. B- and T-cell epitopes of the selected OMPs were predicted using BCPred as well as Propred I and Propred (http://crdd.osdd.net/raghava/propred/), respectively. Each of these OMPs contains regions which are capable to induce B- and T-cell immune responses.ConclusionAnalysis acquired from this study showed that five selected OMPs have great potential for vaccine development against S. flexneri infection. The predicted immunogenic epitopes can also be used for development of peptide vaccines or multi-epitope vaccines against human shigellosis. Reverse vaccinology is a promising strategy for the discovery of potential vaccine candidates which can be used for future vaccine development against global persistent infections.

Functional Characterization of Outer Membrane Proteins (OMPs) in Xenorhabdus nematophila and Photorhabdus luminescens through Insect Immune Defense Reactions.

Xenorhabdus nematophila and Photorhabdus luminescens are entomopathogenic bacterial symbionts that produce toxic proteins that can interfere with the immune system of insects. Herein, we show that outer membrane proteins (OMPs) could be involved as bacterial virulence factors. Purified totals OMPs of both bacterial species were injected into fifth instar larvae of Spodoptera exigua Hübner. Larvae were surveyed for cellular defenses fluctuations in total haemocyte counts (THC) and granulocyte percentage and for the humoral defenses protease, phospholipase A2 (PLA2), and phenoloxidase (PO) activities at specific time intervals. Changes in the expression of the three inducible antimicrobial peptides (AMPs), cecropin, attacin, and spodoptericin, were also measured. Larvae treated with OMPs of both bacterial species had more haemocytes than did the negative controls. OMPs of X. nematophila caused more haemocyte destruction than did the OMPs of P. luminescens. The OMPs of both bacterial species initially activated insect defensive enzymes post-injection, the degree of activation varying with enzyme type. The AMPs, attacin, cecropin, and spodoptericin were up-regulated by OMP injections compared with the normal larvae. The expression of these three AMPs was maximal at four hours post injection (hpi) with P. luminescens OMPs treatment. Expression of the three AMPs in X. nematophila treated insects was irregular and lower than in the P. luminescens OMPs treatment. These findings provide insights into the role of OMPs of entomopathogenic nematode bacterial symbionts in countering the physiological defenses of insects.

Isolation and characterization of outer membrane proteins (OMPs) from Salmonella Gallinarum in chicken and antibiogram of the isolates

Salmonella isolates should be distinguished as it may assist in tracing the source of an outbreak and monitoring trends in antimicrobial resistance associated with a particular type. The specific detection of these Salmonella serotypes is therefore extremely important in order to attribute an isolate to a previously known epidemic outbreak. The present investigation was to isolate and identify S. Gallinarum, to study variation in the profile of outer membrane proteins (OMPs) and to determine in vitro antibiogram of S. Gallinarum in poultry. A total of 228 faecal samples and 22 visceral samples suspected for Salmonellosis were collected, of these 15 samples (6.0%) were found positive for S. Gallinarum. In the present study, rfbS gene sequence was helpful in the serotype-specific detection of S. Gallinarum giving a 187 bp product. Salmonella Gallinarum crude protein extracts determined by SDSPAGE showed migration of OMPs as several bands at approximate moleculer weights of appx. 45 kDa, 55 kDa, 64 kDa, 65 kDa, 74 kDa, 110 kDa, 120 kDa, 135 kDa, 150 kDa,155 kDa, 200 kDa and above 200 kDa. The study indicated a definite variation in the profile of OMPs of various Salmonella Gallinarum strains with major OMPs in the range of appx 80-100 kDa which could be the target for vaccine production. All the isolates tested against 14 antimicrobial agents showed variable susceptibility pattern with highest resistance to nalidixic acid, ampicillin and sulphadiazine and sensitivity to chloramphenicol, gentamicin and enrofloxacin.

Antimicrobial Susceptibility and Characterization of Outer Membrane Proteins of Aeromonas hydrophila Isolated in China

Aeromonas hydrophila isolates from clinical cases (n=43) were tested against 8 antimicrobial agents and typed by outer membrane protein (OMP) pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin (MICs, ≥16 μg mL−1) and sulfamonomethoxine (MICs, ≥64 μg mL−1), but susceptible to norfloxacin (MICs, ≤0.5 μg mL−1). There was a high incidence of resistance to erythromycin (90.70%) and tylosin (93.02%), while a low incidences of resistance to ciprofloxacin (2.33%), enrofloxacin (2.33%) and florfenicol (4.65%). Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective profiles. The strains with the similar OMP profiles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to florfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to florfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to fluoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.

Methionine Mutations of Outer Membrane Protein X Influence Structural Stability and Beta-Barrel Unfolding

We report the biochemical and biophysical characterization of outer membrane protein X (OmpX), an eight-stranded transmembrane β-barrel from E. coli, and compare the barrel behavior with a mutant devoid of methionine residues. Transmembrane outer membrane proteins of bacterial origin are known to display high tolerance to sequence rearrangements and mutations. Our studies with the triple mutant of OmpX that is devoid of all internal methionine residues (M18L; M21L; M118L) indicate that Met replacement has no influence on the refolding efficiency and structural characteristics of the protein. Surprisingly, the conserved substitution of Met→Leu leads to barrel destabilization and causes a lowering of the unfolding free energy by a factor of ∼8.5 kJ/mol, despite the mutations occurring at the loop regions. We report that the barrel destabilization is accompanied by a loss in cooperativity of unfolding in the presence of chemical denaturants. Furthermore, we are able to detect an unfolding intermediate in the Met-less barrel, whereas the parent protein exhibits a classic two-state unfolding. Thermal denaturation measurements also suggest a greater susceptibility of the OmpX barrel to heat, in the Met-less construct. Our studies reveal that even subtle variations in the extra-membrane region of rigid barrel structures such as OmpX, may bear severe implications on barrel stability. We propose that methionines contribute to efficient barrel structuring and protein-lipid interactions, and are therefore important elements of OmpX stability.

Characterization of Outer Membrane Proteins of Escherichia Coli in Response to Phenol Stress

Gram-negative bacteria are generally more tolerant to disinfectants than Gram-positive bacteria due to outer membrane (OM) barrier, but the tolerant mechanism is not well characterized. We have utilized comparative proteomic methodologies to characterize the OM proteins of E. coli K-12 K99+ in response to phenol stress and found that nine proteins were altered significantly. They were OM proteins OmpA, FadL, LamB, and OmpT, cytoplasmic-associated proteins AceA and EF-Tu, inner membrane protein AtpB, putative capsid protein Q8FewO, and unknown location protein Dps. They were reported here for the first time to be phenol-tolerant proteins. The alteration and functional characterization of the four OM proteins were further investigated using western blotting, genetically modified strains with gene deletion and gene complementation approaches. Our results characterized the functional OM proteins of E. coli in resistance to phenol, and provide novel insights into the mechanisms of bacterial disinfectant-tolerance and new drug targets for control of phenol-resistant bacteria.

Isolation and characterization of outer membrane proteins of Edwardsiella tarda and its application in immunoassays

Edwardsiella tarda is an important cause for hemorrhagic septicemia in fish and also for gastro intestinal infections in humans. Sodium lauryl sarcosinate extraction method was used for the isolation of outer membrane proteins (OMPs) from 16 isolates of E. tarda, recovered from snakeheads (Ophiocephalus punctatus). Sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles of OMPs from E. tarda isolates had one major and common protein band of 44 kilodalton (kDa) and 9 other variable protein bands. Rabbit polyclonal antibodies (RPAbs) against OMPs of E. tarda ET-7 detected mainly one common 44 kDa protein in all isolates by western blotting. RPAbs strongly reacted with all isolates of E. tarda by indirect enzyme linked immunosorbent assay (ELISA). These polyclonal antibodies showed no cross reactivity with OMPs or whole cells of other selected aquatic bacteria, i.e., Escherichia coli, Pseudomonas fluorescens, Aeromonas hydrophila, Vibrio cholerae and Flavobacterium ferrugineum. This study indicated that RPAbs against OMPs is useful for detection of E. tarda by ELISA.

Antigenic Characterization of Outer Membrane Protein of Aeromonas sobria Isolated from Goldfish (Carassius auratus L.)

Aeromonas sobria, along with A. hydrophila, has frequently been reported as a causative agent of motile aeromonas septicemia (MAS) in fish and other aquatic organisms. Currently, there are no precise tools to diagnose or vaccinate against this disease. The aim of the present study was to fractionate and characterize the outer membrane protein (OMP) antigen of A. sobria by sero- logical and cellular techniques so as to identify immunoreactive molecules that might be useful in preparing immunodiagnostic and/or immunoprophylactic tools against A. sobria infection in goldfish. Eight fractions were isolated from the crude OMP antigen using sephacryl S-200 and DEAE-cellulose chromatography. The highest seroreactivity was observed in the gel-permeated protein G1 which had an optical density (OD) of 0.72 nm, higher even than that of the crude OMP antigen, 0.63 nm. The serodiagnostic potential of G1 was assessed by using dip-stick ELISA. The in vitro goldfish lympho-proliferation ability of the fractionated antigen, G1A3, was higher than of all the other fractionated antigens and the crude OMP. Therefore, fractionated antigen G1 (molecular wt 42-67 kDa) and G1A3 (molecular wt 45-47 kDa) should be further studied in immunodiagnostic and/or immunoprophylactic preparations for A. sobria infection.

Characterisation of Outer Membrane Protein of Leptospira borgpetersenii Serovar Hardjo

Singh, A., Kumar, M., Kumar, A.A. and Srivastava, S.K. 2005. Characterisation of outer membrane protein of Leptospira borgpetersenii serovar hardjo. J. Appl. Anim. Res., 28: 107–112. Outer membrane protein (OMP) from Leptospira borgpetersenii serovar Hardjo, grown under different cultural conditions, was extracted and characterized for its polypeptide composition and serodiagnostic ability. SDS-PAGE revealed that the detergent insoluble (SI) fraction of OMP from cells grown at the ideal temperature (29C) composed of 5 major polypeptide bands (77, 42.6, 36, 28.8 and 22.5), 4 minor bands (59.5, 47.4, 41.6 and 32 kDa) and 3 very faint bands. In the soluble fraction (SS), different bands were visible, of these 32 kDa and 41 kDa proteins were the most prominant. Cells grown at 22C did not express the minor 18 kDa protein but over expression of 77 kDa was observed. Growth at 38C did not express any additional protein but an enhanced expression of a 31 kDa protein was visible. Change in iron concentration in culture medium did not change the protein profile. Use of OMP as an antigen for conducting ELISA on convalescent animal sera (1:100) revealed that as compared to Microscopic Agglutination Test, it had a Sensitivity and specificity of 100% and 86.4%, respectively.

Characterization of outer membrane proteins in Chlamydia trachomatis LGV serovar L2.

We used a photoactivatable, lipophilic reagent, 3'-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, to label proteins in the outer membrane of elementary bodies of Chlamydia trachomatis LGV serovar L2 and mass spectrometry to identify the labeled proteins. The identified proteins were polymorphic outer membrane proteins E, G, and H, which were made late in the developmental cycle, the major outer membrane protein, and a mixture of 46-kDa proteins consisting of the open reading frame 623 protein and possibly a modified form of the major outer membrane protein.

Identification and characterization of outer membrane proteins of Pasteurella multocida serotype D by using monoclonal antibodies.

Monoclonal antibodies (MAbs) against Pasteurella multocida serotype D were obtained by fusion of spleen cells from BALB/c mice immunized with outer membrane proteins (OMPs) with SP2/0-Ag 14 murine myeloma cells. Desirable MAbs were selected by enzyme-linked immunosorbent assay (ELISA) with OMP as the antigen. MAbs MT1 and MT2 identified two different proteins (H [heavy] and W [weak]), each with a molecular mass of 32 kDa, in Western blots (immunoblots). Treatment of the OMPs with proteolytic enzymes and sodium periodate indicated that the binding sites of MAbs MT1 and MT2 are of protein and glycoprotein natures, respectively. The epitopes reactive with MAbs were surface exposed, as visualized by immunoelectron microscopy. Among field isolates of P. multocida serotype D, two distinct OMP patterns were recognized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and these patterns were designated types I and II. In both the ELISA and the Western blot, MAb MT1 recognized only type I isolates, whereas MAb MT2 recognized both type I and II isolates. Neither MAb MT1 nor MAb MT2 reacted with either reference strains of capsular serotypes A, B, E, and F or field isolates of capsular serotype A of P. multocida. This is the first report of MAbs identifying the serotype D-specific OMP of P. multocida.

Immunochemical and biological characterization of outer membrane proteins of Porphyromonas endodontalis.

Outer membrane proteins (OMP) of Porphyromonas endodontalis HG 370 (ATCC 35406) were prepared from the cell envelope fraction of the organisms. The cell envelope that had been obtained by sonication of the whole cells was extracted in 2% lithium dodecyl sulfate and then successively chromatographed with Sephacryl S-200 HR and DEAE-Sepharose Fast Flow. Two OMP fractions, OMP-I and OMP-II, were obtained, and their immunochemical properties and induction of specific antibodies were examined. The OMP-I preparation consisted of a major protein with an apparent molecular mass of 31 kDa and other moderate to minor proteins of 40.3, 51.4, 67, and 71.6 kDa, while the OMP-II preparation contained 14-, 15.5-, 27-, and 44-kDa proteins as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. OMP-I was found to form hydrophilic diffusion pores by incorporation into artificial liposomes composed of egg yolk phosphatidylcholine and dicetylphosphate, indicating that OMP-I exhibited significant porin activity. However, the liposomes containing heat-denatured OMP-I were scarcely active. Spontaneous and antigen-specific immunoglobulin M (IgM)-, IgG-, and IgA-secreting spot-forming cells (SFC) enzymatically dissociated into single-cell suspensions from chronically inflamed periapical tissues and were enumerated by enzyme-linked immunospot assay. In patients with radicular cysts or dental granulomas, the major isotype of spontaneous SFC was IgG. In radicular cysts, the OMP-II-specific IgG SFC represented 0.13% of the total IgG SFC, while the antigen-specific IgA or IgM SFC was not observed. It was also found that none of these mononuclear cells produced antibodies specific for OMP-I or lipopolysaccharide of P. endodontalis.

Multiresistant serotype O 12Pseudomonas aeruginosa: evidence for a common strain in Europe

A survey was made of serotype association and multiple antibiotic resistance in strains of Pseudomonas aeruginosa in Europe. Of 208 epidemiologically distinct strains from 16 laboratories in 10 countries, 48 were resistant to carbenicillin (MIC greater than 128 micrograms/ml) and gentamicin (MIC greater than 4 micrograms/ml), and 12 of these strains were of serotype O 12. Representative O 12 strains from different countries were compared with two multiresistant O 12 strains isolated 5 years apart, from a British burns unit and the antibiotic sensitive serotype reference strain. All O 12 strains were similar by phage and pyocin typing but lysogenic phage profiles indicated that two strains (the later burns isolate and the serotype strain) were distinct from the others. Electrophoretic characterization of outer membrane proteins, lipopolysaccharides and esterase enzymes corroborated the relationship of the strains and restriction fragment length polymorphism of DNA fragments hybridized with a cDNA probe copy of rRNA from P. aeruginosa provided further proof of their relatedness. We propose that the uniformity of characters of multiresistant O 12 P. aeruginosa in Europe is suggestive of a common origin for the strains.

Antimicrobial susceptibility and characterization of outer membrane proteins of Haemophilus ducreyi isolated in Thailand.

One hundred strains of Haemophilus ducreyi isolated in Thailand from patients with chancroid were tested by the agar dilution method against 10 antimicrobial agents and typed by outer membrane protein pattern by using sodium dodecyl sulfate gel electrophoresis. All strains produced beta-lactamase and were resistant to tetracycline, kanamycin, and sulfonamides. Most had a decreased susceptibility to trimethoprim (MIC for 50% of the strains [MIC50], 0.5 micrograms/ml) and chloramphenicol (MIC50, 8 micrograms/ml). Strains were susceptible to ciprofloxacin (MIC90, 0.001 micrograms/ml), ceftriaxone (MIC50, 0.0015 micrograms/ml), erythromycin (MIC50, 0.015 micrograms/ml), rosoxacin (MIC50, 0.03 micrograms/ml), and spectinomycin (MIC50, 8 micrograms/ml). The degree of antimicrobial resistance found in Thailand is higher than that reported for H. ducreyi isolated in other regions. Five different outer membrane protein patterns were found by analyzing proteins in the range of 29 to 61 kilodaltons, but 98% of the Thai strains fell into three patterns which did not differ greatly. Outer membrane protein patterns of Thai strains were also seen in strains from other geographic areas. A new outer membrane protein type was found among nine strains isolated in Singapore.

Identification and characterization of Campylobacter jejuni outer membrane proteins.

Outer membrane proteins from isolates of Campylobacter jejuni were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sarcosinate-insoluble membrane preparations were outer membrane enriched based on increased ketodeoxyoctonate concentrations, the presence of surface-exposed 125I-labeled proteins that were hydrophobic, and similarity to membrane vesicle (bleb) sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. Most isolates contained a single major band with molecular weight of 41,000 to 45,000. Profiles of C. jejuni and Campylobacter coli isolates were indistinguishable, but either could be easily differentiated from Campylobacter fetus and Campylobacter faecalis. The profiles were stable for strains under a variety of growth, incubation and passage conditions. We classified 110 isolates from patients with sporadic campylobacter enteritis into nine subtypes based on differences in outer membrane sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles. Two categories accounted for 76% of the isolates. Complete concordance was observed in subtypes of strains obtained from epidemiologically related cases. Thus, comparison of the major outer membrane proteins of C. jejuni is a useful technique for investigating the transmission of this organism and may provide a basis for immunological characterization of the outer membrane proteins.